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Diagnostic Immunohistochemistry: What can

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 REVIEW ARTICLE
Diagnostic Immunohistochemistry: What can Go Wrong?
Hadi Yaziji, MD* and Todd Barry, MD, PhDw
Abstract: Although immunohistochemistry (IHC) has become
an integral component of the surgical pathology laboratory and
practice, it remains a subspecialty in search of a definition. The
rapid expansion of this discipline is continually contributing to
the struggle of interested pathologists and technologists to
identify the proper working conditions of antibody reagents and
the right choice of detection systems and instrumentations.
Training programs have not, for the most part, successfully
incorporated IHC in their curricula. Thus, pitfalls in diagnostic
IHC, in our opinion, are becoming more frequent than they used
to. In this review, we highlight and attempt to provide solutions
for the most common pitfalls in diagnostic IHC as it relates to
the routine practice of surgical pathology.
Key Words: immunohistochemistry, pitfalls, immunophenotype,
antibodies, antigens, detection systems, false-positive, false-
negative, endogenous biotin, immunoreactivity, HIER, antigen
retrieval
(Adv Anat Pathol 2006;13:238246)
T he role of immunohistochemistry (IHC) and ancillary
technologies is critical to the practice of surgical
pathology. Historically, the integration of IHC into the
daily surgical pathology practice has been slow and
unceremonious. In the late 1980s and early 1990s, most
pathology practices were introduced to IHC with only a
limited panel of markers (ie, vimentin, S-100, k, l). The
gradual and steadfast expansion of the antibody reper-
toire has taken almost 2 decades, and now approaches
several hundred antibodies that have been validated for
clinical use (in vitro diagnostic) and are available for
diagnostic use. During most of this period, IHC has been
regarded primarily as an ancillary test, analogous to a
special stain.
This steadfast expansion of IHC has been accom-
plished by considering IHC as an extension of special
stains. In a similar fashion, most residency training
programs have not incorporated comprehensive and
structured instruction of IHC into their curricula. To
date, many still do not view IHC as a distinct discipline in
pathology [like gastro-intestinal (GI) pathology, hemato-
From the *Ancillary Pathways, Miami, FL; and wPhenoPath Labora-
tories, Seattle, WA.
Reprints: Hadi Yaziji, MD, Ancillary Pathways, LLC, 7533 SW 58th
Avenue, Miami, FL 33143 (e-mail: yaziji@ancillarypath.com).
Copyright r 2006 by Lippincott Williams & Wilkins
238
pathology, dermatopathology, etc], but as a simple
technique.
As shown in Table 1, pitfalls in the use of IHC can
occur at the technical, or design and interpretation phases
of work-up. Therefore, technologists, in addition to
pathologists, should be aware of these pitfalls. Ideally,
the technologist and the pathologist should actually work
together in the validation, testing, design, and interpreta-
tion of IHC assay. The benefits of such ideal working
relationship are immense.
PITFALLS AT THE TECHNICAL LEVEL
Significant similarities exist between IHC and
cooking. In the kitchen, we select ingredients, the manner
in which these ingredients are cooked (ie various heat
sourcescharcoal, gas, electric, steamer, pressure coo-
ker), what type of sauce to add and the way we mix
everything together into a delicious dish. Similarly, in the
laboratory, proper fixation of the specimen, proper
cutting of tissue sections, selection of the proper antibody
clone and buffer as well as the type of retrieval
(microwave, steam, water bath) are key to a successful
antibody immunostaining.
Specimen Fixation
It thus should not come as a surprise that pitfalls in
diagnostic IHC begin with the way we fix our specimens.
In fact, antigen degradation does occur even before tissue
fixation. Fresh specimens that are subjected to prolonged
drying before fixation can suffer significant loss of
antigenicity. With the increasing awareness of the harmful
effects of formaldehyde and increasing difficulty with its
disposal, many new formaldehyde alternatives are now
commercially available and have replaced formalin in a
small number of anatomic pathology laboratories. Most
of these fixatives are glyoxyl-based. The problem with
many of these fixatives is that they may render the tissue
unsuitable for IHC methods. Examples include the drastic
reduction of CD5 by B5 fixed tissue1 and the compro-
mised level of hormone receptors and Ki-67 in glyoxyl-
fixed tissue. The latter has equally disastrous effects on
fluorescence in situ hybridization.2 Formaldehyde, despite
its documented toxic effects, remains, to date, the favored
fixative of all specimens especially those requiring
hematopathology immunophenotyping.
The length of tissue fixation also has significant
effects on immunoreactivity. To paraphrase Dr Hector
Battifora, all tissues received in routine pathology
laboratories are either under-fixed or over-fixed in
formalin, that is, none are optimally fixed for antigenic
Adv Anat Pathol ! Volume 13, Number 5, September 2006
Adv Anat Pathol ! Volume 13, Number 5, September 2006
TABLE 1. Pitfalls in IHC can Occur at 2 Main Levels
Technical Pitfalls
Fixation
Processing
Pre-treatment
Detection System
Professional pitfalls
Selection of panel
Sensitivity of panel
Choice of antibody type
Choice of antibody clone
Interpretation of results
Interpretation of literature
retention. However, in this postepitope retrieval era, over-
fixation of tissue (by formalin) may not necessarily be as
detrimental to antigenicity as previously thought. For
example, Poston et al3 have demonstrated the expression
of Hodgkin-related antigens in the original tissue
obtained by Sir Thomas Hodgkin 150 years ago. It is
usually easier to overcome the problems of over-fixation
than under-fixation. The latter is often an unfortunate
consequence of time pressures to minimize turnaround
time in pathology laboratories, despite the fact that we
generally have no control over what time in the day
tissues are obtained, and in what form the tissues are
received. Formalin takes more than a few hours to
adequately fix even small pieces of tissues,4 and it is not
uncommon for tissues arriving in an unfixed state in the
surgical pathology laboratory at 5:00 PM to be cut and
placed in a tissue processor where the total immersion
time in formalin will be less than 2 or 3 hours. In these
situations, because the next step in the tissue processor is
invariably immersion into alcohol (which, in contrast to
formalin, fixes rapidly), late arriving tissues may be amplification methodology, also referred to as the
predominantly alcohol rather than formalin-fixed; worse,
they are partially alcohol and partially-formalin fixed,
which is particularly problematic for some antigens. The
same can be said for cell blocks of body fluids, which may
be processed in formalin once spun down and pelleted,
but the cells may have sat in an alcohol-based solution
before cell block processing. Similarly, under-fixed tissues
exhibit uneven IHC signal as edges may be better fixed
than the interior of tissue.
The use of fixatives that have traditionally been
used for special tissues, such as B5 fixative for bonds for calcium ions.4,9 Alterations in the 3-dimen-
lymphoid tissues and Bouin fixative for gastrointestinal
tract biopsies is a practice for which there is no longer
good justification. Both of these fixatives manifest more
restricted antigen preservation than formalin, and the use
of these fixatives not infrequently results in selective
antigenic loss that can be annoying at best and
catastrophic at worst in the work-up of a given case.
Tissue Processing
It is also critical to follow strict procedural steps
before incubating the primary antibody on the tissue
section, to avoid detrimental staining results. For
instance, paraffin embedding at temperature in excess of
r 2006 Lippincott Williams & Wilkins
Diagnostic Immunohistochemistry
601C can compromise some of the epitope binding sites.
Microtomy sectioning of sections thinner than 3 mm or
thicker than 7 mm will lead to suboptimal results. Thinner
sections will result in a very weak signal and thicker
sections will lead to either loss of tissue section from the
glass slide or great difficulty in evaluating a thick section.
Detection Systems
The choice and application of detection system are
equally important. The current widely used detection
system is the avidin biotin immunoperoxidase technique5
in which a primary unlabeled antibody is detected using a
secondary biotin-conjugated antibody from a different
species; detection is possible through a biotinylated
peroxidase which is incorporated into a complex
assisted by an avidin, which acts as a molecular glue
binding the biotinylated secondary antibody to the
biotinylated peroxidase. A novel innovation in recent
years has been the polymer-based detection systems.6,7 As
with the avidin biotin immunoperoxidase technique, an
unlabeled primary antibody is first employed, but in this
2-step procedure, the second reagent is a dextran polymer
to which are bound large number of peroxidase molecules
and secondary antibody molecules to bind to the
unlabeled primary antibody. As will be noted below, this
system has the advantage of being avidin-biotin-free,
which eliminates the nonspecific biotin signal that can
result in nonspecific staining in some tissues, particularly
liver and kidney. A number of polymer detection systems
have recently become available.
Although the avidin biotin and dextran-polymers
immunohistochemical methods are currently state-of-
the-art secondary reagents for diagnostic IHC, the next
generation IHC technique involves a tyramine-based
catalyzed reporter deposition or catalyzed system
amplification method.8 These new detection systems can
provide a highly sensitive method of detecting antigens
that may be present in low levels and thus, offer some
advantages in this setting.
Tissue Pretreatment
Formalin fixation is thought to act on tissues
through the formation of protein-protein cross-links,
presumably with the additional formation of coordinate
sional structure of proteins resulting from cross-linking
change the 3-dimensional configuration of the native
antigen, altering antibody-antigen interactions. The
introduction of epitope retrieval techniques in the early
1990s revolutionized the practice of diagnostic IHC by
focusing attention on the undoing protein cross-
linking. Shi and colleagues,10 by placing tissues in a
microwave oven in a buffer, found a remarkable
enhancement of immunostaining using 39/52 different
antibodies tried, including antibodies to wide range of
different antigens. Cattoretti et al11 also showed that new
antibodies such as MIB-1, seemed to require microwave
pretreatment of the tissue for the antibody to work on
239
Yaziji and Barry
fixed tissues. Several comprehensive studies of micro-
wave-based epitope retrieval have been reported, with
similar results.1214 Nonetheless, excessive tissue micro-
waving can destroy antigenicity, and, reflecting gradients
in fixation, there can result gradients of immunoreactivity
in the tissue. More commonly, areas of loose connective
tissue and fat can display a very characteristic micro-
wave burn pattern. Techniques employing sources of
heat other than microwaves have been introduced. These
include: autoclaving15; high temperature water bath;
pressure cooking16; and steam heating.17 Selection of
the pretreatment step may have a large impact upon the
final immunostained result.18 The combination of heat
source, enzyme digestion, buffer type, and time of heating
can produce drastically different results. At our institu-
tions, when we acquire a new antibody, we experiment
with >3 types of pretreatment combinations along with
>3 different dilutions until we achieve an optimal signal.
Pretreatment instructions included in antibodies package
inserts should be used as a guide, but they do not always
offer the best end result.
PITFALLS AT THE DESIGN AND
INTERPRETATION LEVEL
Selection of Panel
The optimal goal of IHC techniques in the diagnosis
of surgical pathology is to maximize sensitivity without
compromising the specificity of the study results. To
achieve a maximum sensitivity of target detection, it is
always a good practice to use more than one antibody for
a given antigenic target. For instance, if one suspects
neuroendocrine phenotype in a carcinoma, one could use
a combination of synaptophysin and chromogranin. It is
often (albeit not too common) that the lesion will be
positive with one or the other, but not both. In that
respect, there should be no utility for neuron-specific
enolase in the modern IHC laboratory. This marker has
proven its lack of specificity over the years. Another
example of the importance of selecting antibodies in a
panel relates to screening studies for undifferentiated
malignant neoplasms. The most common panel includes
cytokeratin, vimentin, S-100, and CD45, with the
perception that it will allow the pathologist to discrimi-
nate (with high level of sensitivity) between carcinomas
(keratin positive), sarcomas (vimentin positive), lymphomas
(CD45 positive), and melanomas (S-100 positive). In reality,
vimentin is positive on many carcinomas (thyroid, renal cell,
any carcinoma with spindle cell features),1922 mesothelio-
mas, and lymphomas.23 It is negative on some sarcomas (ie,
alveolar soft part). Cytokeratin should be absent on adrenal
cortical carcinomas24 and positive on many sarcomas
(especially leiomyosarcomas)2529 and melanomas.24,30 S-
100 is undoubtedly absent on a subset of melanomas (about
2% to 10%) and positive on many carcinomas (particularly
thyroid, salivary gland, and renal cell). CD45 is also absent
on some lymphomas (classic Hodgkin) and leukemias.
A more realistic panel should include cytokeratin
(for carcinomas), CD43+CD45 (which will cover most
240
Adv Anat Pathol ! Volume 13, Number 5, September 2006
TABLE 2. Conventional and Preferred Panel for the Work-up
of Undifferentiated Malignant Neoplasms
Diagnostic
Category Conventional Panel Preferred Panel
Carcinoma Pan-cytokeratin Pan-cytokeratin
Lymphoma CD45 CD45+CD43
Melanoma S-100 S-100 and either gp100 or
MelanA
Sarcoma Vimentin Collagen type IV and vimentin
lymphomas, leukemias, and anaplastic plasmacytomas) and
2 melanocytic markers, S-100 and either gp100 (recognized
by clone HMB-45), or melanA, and collagen type IV and
vimentin as sarcoma markers (Table 2). It has been our
experience and others31 that the consistent expression of
collagen type IV around spindle cells in mesenchymal
tumors makes this marker useful in this differential
diagnostic setting.
The Design of Panel: Algorithm
It is very important to emphasize the algorithmic
approach in the IHC work-up of any lesion, namely
starting with a limited but highly sensitive panel, and
expanding with more specific markers depending on the
results of the primary panel, as seen in Table 3 below.
A haphazard selection of markers is almost a guarantee
for missing the correct diagnosis (unless one empties the
refrigerator and randomly applies >20 antibodies).
This is particularly true if the pathologist has not
included a proper differential diagnosis for the IHC work-
up of the tumor in question. For example, if one does not
consider the diagnosis of granulocytic sarcoma (chlor-
oma) in the differential diagnosis, it practically does not
matter how many markers are applied unless such
markers as CD43, CD34, CD117 (c-kit), myeloperoxidase
(and others) are part of the panel.32 Other lesions that we
have seen consistently been under-recognized are breast
angiosarcomas and metastatic melanomas to solid organs
and central nervous system (because of their epithelioid
morphology), as listed in Table 4. To paraphrase our
mentor, Dr G. Randolph Schrodt, in pathology, chance
also favors the prepared mind.
A wise and safe approach would be to start the
panel with the most general diagnostic categories in mind
and then to narrow the scope of the IHC panel to more
specific antibodies based on the results of the initial panel.
TABLE 3. Algorithm Approach in the Design of IHC Panels
Algorithm Component Description
Step 1: maximum Start with a panel that offers the most
sensitivity sensitive and comprehensive results
Step 2: maximum Apply more specific markers to confirm
specificity the results obtained in step 1
Step 3: final diagnosis May not be necessary if step 2 work-up
offered definitive results
Usually accomplished with 1 or 2
markers
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Adv Anat Pathol ! Volume 13, Number 5, September 2006 Diagnostic Immunohistochemistry

low sensitivity of calretinin in detecting mesothelio-

TABLE 4. Frequently Encountered Misclassified or Under-
recognized Lesions in our Practices ma.35 The authors used a calretinin clone that should
have been discontinued and neglected to use the more
Organ Entity Mistaken asy
widely used clone. Unfortunately, the pathology literature
Lymphoid Granulocytic sarcoma PD Carcinoma in the past 15 years is cluttered with such false claims of
(nodes, BM, etc)
CD30-negative Hodgkin Anaplastic large cell
either superior or poor antigens, whereas in reality it is
Breast Angiosarcoma Carcinoma the choice of a given antibody clone (and not the antigen)
Sclerosing adenosis Invasive carcinoma that is the source of such claim. Selection of the
Any organ Melanoma Carcinoma appropriate clone can have a large impact on the
detection of a given antigen.
In some situations, there is yet to exist a reliable
For example, if a pathologist faced with a pleural-based antibody clone. A common pitfall in the selection of such
epithelioid tumor designs a panel of markers directed that markers includes antibodies to CA-125. This is arguably a
address the differential diagnosis of carcinoma of useful serum marker that guides the oncologists to search
unknown primary site, he may miss the diagnosis of for and detect ovarian tumors; however, the tissue-based
mesothelioma altogether. Thus, the initial panel chosen equivalent has a very compromised specificity. In our
should be broad in scope, first excluding mesothelioma experience, it has been positive on GI and breast tumors.
and establishing the diagnosis of carcinoma and then We do not offer CA-125 antibody test in our respective
proceeding to a more focused work-up of a lung primary laboratories. An excellent alternative is WT-1 antibody
versus metastatic carcinoma to the pleura. for the detection of ovarian serous tumors.36
Using the principle of maximum sensitivity and
specificity, one should always attempt to fine tune Specific and Nonspecific Immunoprofiles
existing panels to address varying differential diagnosis Sometimes, it is our lack of knowledge that certain
considerations, not to mention that such panels must be lesions exhibit specific and reproducible immunoprofiles
designed in the context of morphologic and clinical that hinders our ability to use IHC to confirm (or refute)
features of a given tumor. On occasion, the clinical the diagnosis of these lesions. For instance, the combina-
information offers the most sensitive and specific data to tion of CK+, MOC31 " , CD10+ (in a bile canalicular
help guide selection of an antibody. A 67-year-old woman pattern), and CD34+ (in a sinusoidal pattern) profile is
with a history of breast cancer and a recently discovered virtually diagnostic of hepatocellular carcinoma. Table 5
tumor in the lung most likely has either metastatic breast contains a list of entities with specific immunophenotypes.
cancer or primary lung cancer, and there is no need to Conversely, expression of the cdx-2 GI transcription
include GI and ovarian markers in the primary panel, factor is not at all specific for GI tumors. As we and
which could be limited to mammary and lung markers
(TTF-1, surfactant, ER, PR, GCDFP-15, or mammaglo-
bin). If the results of the primary panels are not definitive,
TABLE 5. Examples of Pathologic Entities With Unique
then one can apply a secondary panel of markers that Diagnostic Immunophenotypes
should cover other solid organs.
Pathologic Entity Unique Immunophenotype
Importantly, there is always a continual evolution
of antibodies over time. Markers that were once Hepatocellular CD34 in a sinusoidal pattern, collagen
considered state-of-the-art are eventually replaced by carcinoma type IV around tumor nests
Meningioma Progesterone receptor, EMA
markers that prove to be more sensitive and specific. For Hemangioblastoma S-100, EGFR
instance, a panel of antibodies used in the differential Barrett esophagus CDX-2 on intestinalized cells
diagnosis of adenocarcinoma versus mesothelioma once Hydatidiform mole P57 (for complete moles), FISH (for triploid
included antibodies such as vimentin, carcinoembryonic partial moles)
Hairy cell leukemia CD20, CD25, TRAcP, cyclin D1
antigen, epithelial membrane antigen, and B72.3. How- Fibromatosis B-catenin (nuclear signal), smooth muscle
ever, more sensitive and specific markers now can be used actin (not desmin)
in this differential. Positive carcinoma markers should Glomus tumor Smooth muscle actin (not desmin),
include MOC-31, BG8, and BerEP4 (as epithelial synaptophysin
glycoproteins) while positive mesothelioma markers Endometrioid Vimentin, B-catenin (nuclear)
carcinoma
should include calretinin, WT-1, cytokeratin 5, and PML SV40, p53 (invariably positive)
mesothelin.33 It is usually sufficient to establish the Monophasic synovial Keratin, CD99, b catenin
diagnosis with only 2 epithelial and 2 mesothelial sarcoma
markers. The recently described lymphatic endothelial PEComa Smooth muscle actin, gp100 (HMB-45)
Kaposi sarcoma HHV8
marker podoplanin seems to be highly sensitive and Follicular dendritic cell CD21, CD35, CD23
specific mesothelial marker.34 tumor
Inflammatory MFB Smooth muscle actin (filamentous pattern),
The Choice of Antibody Clone tumor EBV, ALK-1
Not all antibody clones are created equal. A typical Merkel cell tumor Ck20-positive (dotlike), synaptophysin-
positive, TTF-negative
example is a study published in 2001 that described the

r 2006 Lippincott Williams & Wilkins 241

Yaziji and Barry
others documented,37 cdx-2 is expected to be positive on
ovarian mucinous tumors and bladder adenocarcinomas
because of the GI phenotype of these tumors. Not to
forget the primary mucinous lung carcinomas with GI
(TTF " , surfactant " , cdx2+) phenotype.38 These are
usually adenocarcinomas with partial bronchioloalveo-
lar/mucinous features. Also, as mentioned above, mela-
nomas and leiomyosarcomas can often express
cytokeratin.39,40 This observation emphasizes the danger
of using a single marker to attempt to establish a
diagnosis.
Another common pitfall, we encounter in our
practice is the reflexive designation of CD30+/CD15 "
tumor as anaplastic large cell lymphoma. It is not
uncommon to receive a request from a colleague to
perform p80/ALK-1 studies to confirm anaplastic large
cell lymphoma on tumors with the above results. With
minimal work-up (absence of CD3, CD43), one can
suspect the possibility of CD15-negative Hodgkin lym-
phoma, not to mention the more common diagnosis of
CD30-positive diffuse large B-cell lymphoma, which can
be established by showing expression of CD79a (and/or
CD20), and coexpression of CD45 (along with absence of
CD3, CD43, and ALK1). Similarly, one could easily
make a misdiagnosis of grade I follicular lymphoma
simply on the basis of BCL-2 expression on the lymphoid
follicles. Resting or primary follicles by definition do not
have germinal centers and thus are composed primarily of
BCL-2+ mantle zone B cells, which can impart the
impression of a BCL-2+ follicular neoplasm. However,
these cells show prominent IgD positivity, characteristic
of resting follicles that are not typical of follicular
lymphoma.
Specific knowledge of the right expression (staining)
pattern of a given marker is also crucial. As noted above,
the most common (and costly) error in interpretation of
markers that we have observed over the years is false-
positive interpretation of endogenous biotin. A review of
the etiologies of false-positive signal is in order: Gen-
erally, when all cells and tissues appearing positive on a
given slide is caused by excess antibody concentration, an
example of nonspecific signal. A general rule of thumb distribution of CD15 and CD30 in Hodgkin cells), and
is that antibodies should be used on tissue sections at a
protein concentration of approximately 1 mg/mL (or less).
However, some can tolerate higher concentrations, and
others will start nonspecifically sticking to tissue at even
2 or 4 times the recommended concentration. Some
antibodies (eg, HMB-45) will appear to react with all cells
in tissues as a function of inappropriate fixation (eg, B5
fixed tissue). As has been noted, the distinction of
immunostaining (which can be artefactual) and expres-
sion is an important one.41 False-positive immunostain-
ing can be caused by the use of antibodies at
inappropriately high concentrations,42 potentially altered
specificities following the use of epitope retrieval techni-
ques,43 and the cross-reactivity of anticytokeratin anti-
bodies with other proteins.44 Another very common
reason for false-positive immunostaining is the adverse
effects of avidin biotin-based IHC detection systems:
242
Adv Anat Pathol ! Volume 13, Number 5, September 2006
there is endogenous biotin present in many tissues
(particularly liver and kidney), and also many tumors.
The latter tissues, in particular, can yield false-positive
signals with avidin biotin-based techniques, as amplifica-
tion of the endogenous biotin can be just as effective in
yielding a brown (or black, or red, etc.) signal as can the
binding of the primary antibody to its target antigen.
Usually, endogenous biotin signal can be distinguished by
its fine, granular cytoplasmic pattern; while this can help
if the primary antibody is expected to yield a nuclear
signal, if the antibody being employed is expected to yield
a granular cytoplasmic pattern (eg, antibodies to chro-
mogranin A), this can pose a serious problem. In addition
of using of biotin block steps to avoid this problem, we
strongly advise the use of alternative, avidin and biotin-
free detection systems such as the polymer-based
reagents. We favor the latter.
The issue of false-positive immunostaining cannot
be dismissed as only a function of inappropriately high
antibody concentrations or other factors (eg, biotin)
unrelated to the primary antibody. Antibodies may, in
fact, stick to tissue by mechanisms other than antigen-
antibody binding, and these can be some of the most
difficult artifacts to identify. Examples of undesired
specific signal include the apparent specificity of many
different antibodies for pancreatic islet cells, the stick-
ing of antiprogesterone receptor antibodies to mucin
globules, and the nuclear signals sometimes seen with the
L26 anti-CD20 antibody. Help in distinguishing true,
desired immunostaining from true, undesired immuno-
staining is often found by noting the intracellular
localization of the signal.
Antibodies define antigens, which have, in general, a
well-characterized locus and pattern of expected signal.
There are antigens which are exclusively nuclear (eg, TdT,
TTF-1, estrogen receptor), antigens exclusively cytoplas-
mic, (eg, cytokeratins, thyroglobulin, surfactant ApoA),
antigens with combined nuclear and cytoplasmic localiza-
tions (eg, S-100 protein, calretinin), antigens localized to
the cell membrane (eg, CD45, HER-2/neu), antigens with
complex distributions (eg, the membranous plus Golgi
antigens with exclusively extracellular localization
(eg, type IV collagen).
Different antibodies often yield characteristic pat-
terns in addition to characteristic localizations within
cells. For example, antibodies to S-100 produce a
homogenous signal; antibodies to cytokeratins can
produce a filamentous signal; and antibodies to chromo-
granin A generally produce a granular signal. (These
patterns in part reflect the subcellular distribution of the
antigen in question, eg, the localization of chromogranin
A to discrete dense core granules.)
Different antibodies also can yield characteristic
patterns across tissues or tumors. For example, antibodies
to nuclear transcription factors such as TTF-1 and CDX-
2, in lung carcinomas and colorectal adenocarcinomas,
respectively, when positive on a given tumor, are positive
in most tumor cells, whereas markers to proteins of
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Adv Anat Pathol ! Volume 13, Number 5, September 2006
terminally differentiated cells such as the gross cystic
disease fluid protein-15 of breast cancers or uroplakin of
urothelial carcinomas, are generally positive in only a
small fraction of the tumor cell population.
All these factors (the specificity of the antibody, its
subcellular distribution, its pattern of reactivity within a
given cells and its pattern of reactivity within a popula-
tion of cells, together with known possible undesired
specific immunostaining tendencies), constitute an anti-
body personality profile, which is something generally
learned with experience and often not provided in the
package insert. Part of the antibody personality profile,
however, is a multidimensional matrix of fixation factors,
antibody concentration, tissue pretreatment, and detec-
tion systems, which are the optimal operating space for B-cell marker.
a given antibody. If you are inadvertently operating
outside of this optimal operating space, that is, in a region
of inappropriately high antibody concentration or in-
appropriately fixed tissue, a false-positive signal may
result.
Degree of Expression is Contextual Interpretation Aspects
Sometimes expression of a marker on a subset of
tumor cells is as insignificant as no expression at all and
sometimes these findings are just the tip of the iceberg in a
specimen that may be poorly fixed. For instance,
expression of any of the intermediate filament cytoke-
ratins on a few tumor cells in a given carcinoma can, with
few exceptions (ie, small cell carcinomas) be dismissed as
negative. A metastatic carcinoma to the liver with a
uniform cytokeratin 20 signal and only rare cells positive
for cytokeratin 7 signal should not be labeled as
cytokeratin7+/20+ tumor. Conversely, if a metastatic
carcinoma that shows coexpression of cytokeratins 7 and
20 on >20% of tumor cells also exhibits a weak and
focal cdx-2 signal, the latter should not be dismissed as
negative. Low levels of cdx-2 are not uncommon among
pancreatic and gastric carcinomas. The only GI tumors
that almost always express high levels of cdx-2 are those
of colorectal origin.37
Scoring of a signal as positive or negative is not only
based on the statistical probability of significance of such
signal, but sometimes on the clinical meaning of various
levels of expression. In the field of prognostic factors,
expression of estrogen receptor on >1% of a breast
cancer cells has been shown to be associated with clinical
response to tamoxifen.45 On the other hand, even in
tumors with high levels of estrogen receptor expression,
absence of progesterone receptor expression has been
shown to predict a poor outcome, hence the significance
of testing for both receptors at the same time.46
SPECIAL SITUATIONS: IHC IN
HEMATOPATHOLOGY
Technical Aspects
Perhaps the most important technical problems
related to IHC in hematopathology are the use of
nonformalin fixatives on bone marrow and lymph node
r 2006 Lippincott Williams & Wilkins
Diagnostic Immunohistochemistry
specimens. For instance, cyclin D1 and CD30 can be
severely compromised in B5-fixed tissue.47 Decalcification
has long been known for having negative impact on the
tissue antigenicity.48,49 Not to mention the same issues
related to general diagnostic IHC would also apply to this
topic, including suboptimal epitope retrieval and impro-
perly titered antibodies. What is also unique to lympho-
proliferative disorders is the frequent occurrence of target
antigens expressed at low levels. Examples include low
levels of CD2050 and ZAP-7051 in CLL/SLL. Another
common pitfall especially in the work-up of previously
treated B-cell lymphomas with anti-CD20 Rituxan is the
virtual elimination of CD20 signal as a result of
treatment.52 CD79a would be one good alternative as a
Artifactual staining can sometimes be a source of
interpretation problems, such as the deposition of
mercury in B5-fixed tissue.47,53 As mentioned above,
many are familiar with the specific and reproducible
nucleolar CD20 signal on non-neoplastic cells.
Table 6 below summarizes the different patterns of
antigen localizations in lymphoid lesions. As we discussed
above, lack of knowledge of subcellular localization of a
given antigen may lead to problems in correctly inter-
preting results.
Perhaps, the least known pitfall in diagnostic IHC
of hematolymphoid disorders is the infidelity of tumor-
specific markers. Table 7 highlights a list of potential
anomalous expression of various markers that have been
well documented in the literature. For example, CD20
expression has been reported in a subset of peripheral T-
cell lymphomas.54 Also, BCL-2 is negative in a subset of
follicular lymphomas, especially those of higher grade.55
Another common problem in diagnostic IHC of
lymphomas is the difficulty in demonstrating light chain
restriction. It is usually easier to highlight either k or l
light chain restriction in neoplasms with plasmacytoid
differentiation.56 However, there are occasional circum-
stances in which light chain restriction may not be
sufficient for the diagnosis of lymphoma. This pheno-
menon has been described in Castleman disease,57 and in
marginal zone hyperplasia.58
Finally, the use of ancillary molecular tools is
always helpful in assessing B or T-cell clonality (by
performing immunoglobulin heavy chain and T-cell
receptor gene rearrangement, respectively). Furthermore,
TABLE 6. Localization of Various Hematolymphoid Markers
in Diagnostic IHC
Localization Examples
Nuclear BCL-6, PAX-5, OCT-2, BOB-1
Nuclear+cytoplasmic BCL-10
Cytoplasmic BCL-2
Membranous CD20, CD3, CD5, CD23, CD43, CD79a, etc
Membranous+Golgi CD15, CD30
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Yaziji and Barry
TABLE 7. Tumor-specific Markers and Their Infidelity
Examples
Marker(s) Example
CD20 Expression in PTCLs
CD79a Expression in T-ALL
TdT Expression in AML
CD30 and CD30+/CD15 " in 10-15% of classic
CD15 Hodgkin
CD30+/CD15 " in ALCL
CD30 and CD15 expression in diffuse
large BCL
CD30 and CD15 expression in PTCL
BCL-2 Expressed in mantle zones of primary
follicles
Negative in a subset of follicular
lymphomas
B-cell markers Expressed in classic HL
PAX-5 can be seen in NE tumors
CD138 Can be expressed in epithelial tumors
ALCL indicates anaplastic large cell lymphoma.
identification of specific translocations either by PCR or
more recently by fluorescence in situ hybridization has
also shown its utility in the routine of hematopathology
diagnosis of hematolymphoid neoplasms.
LIMITATIONS OF IHC
Table 8 shows a list of diagnostic entities where IHC
should play a limited role. As an example, one of the
common requests that we receive is to perform IHC
studies to distinguish between primary squamous cell
carcinoma of the lung and metastatic squamous cell
carcinoma from the head and neck. These 2 types of
tumors are immunophenotypically identical. The only
subtypes of squamous carcinoma that show some
discriminating features are nasopharyngeal carcinoma
(positive for Epstein-Barr virus genome59,60) and cervical
squamous carcinoma (sometimes positive for estrogen
receptor). Similarly, both skin appendage tumors and
squamous cell carcinomas of the skin can show similar,
if not identical immunophenotypes.
LITERATURE SEARCH
In addition to Medline, Immunoquery has become
an increasingly popular Internet website and the top
search engine of choice for many pathologists seeking
specific information on IHC. Although the editors of that
TABLE 8. Examples of Diagnostic Entities Where IHC Still
has a Limited Role
Distinction between squamous carcinomas of various organs
Prediction of stromal invasion in cervical and vaginal squamous
intraepithelial lesions
Distinction between DH, ADH, and DCIS in the breast
Assessment of stromal invasion in cutaneous squamous carcinomas
Distinction between skin appendage tumors and primary squamous or
basal cell carcinomas
244
Adv Anat Pathol ! Volume 13, Number 5, September 2006
website (Immunoquery) are to be commended for their
efforts, we do not recommend using it as the main source
of information. This is because the site combines the
References
literature on any given subject together as if all the studies
52 performed on that subject are of equal quality. Moreover,
54
54
different Ab clones may be used in different studies and
55 the definition of positivity may be defined differently in
various studies, making comparison of the studies next to
56, 57 impossible. The reality is that the data gleaned from this
58, 59 website may not be very helpful at best and misleading in
51, 59 some instances.
53
HOW TO MINIMIZE THE ERRORS IN IHC
60
At the technical level, no antibody should be
acquired without the basic knowledge of its performance
characteristics and the expected expression (staining)
pattern. If possible, the avidin-biotin detection system
should be replaced by a polymer-based one to minimize
the sources of false-positive endogenous biotin signal.
More importantly, pathologists should be working closely
with the technologists to educate one another about the
various issues discussed in this material. The chance for
things going wrong in the IHC laboratory is real and not
a perceived one, and only a skilled technologist can
function as the gatekeeper with the ability to identify and
troubleshoot various problems in the laboratory.
It is very difficult, even for an experienced patho-
logist, to keep up with the literature on new and existing
markers, making the field of IHC one of the fastest
growing and more exciting subspecialties to study in
pathology. Therefore, pathology groups should have at
least one member with the expertise and interest to be
responsible for the day-to-day laboratory functions and
the interpretation and design of IHC panels.
The algorithmic approach of highly sensitive
screening panels followed by highly specific diagnos-
tic panels should be adopted. This practice is safe and
constitutes good medicine. After all, the main goal of
performing IHC studies is to strengthen and extend our
hematoxylin and eosin (H&E) diagnostic power. The IHC
literature should always be interpreted with caution and
at least some degree of skepticism. Moreover, it is
important to gain experience with particular antibodies
and continually compare your experience with that of the
established literature.
Residency training programs that have not fully
incorporated IHC in their curricula, should do so with the
help of experienced pathologists. The highly expensive
IHC reagents and their availability make it practically
impossible for any pathology to be self-taught without the
proper environment.
In summary, the same guiding common sense
principles that a great pathologist applies to his/her
approach to H&E diagnoses can be applied to the
practice of IHC. Those of us who make fewer errors in
their conventional H&E practice will likely be making
fewer errors when dealing with IHC.
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Adv Anat Pathol ! Volume 13, Number 5, September 2006
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